To summarize the results from the current study, gadolinium at concentrations up to 50 µM had no effect on proliferation or differentiation under basal conditions (i.e., in SMEM-dFBS) in any of four human colon epithelial cell lines. However, when the calcium concentration was increased to 1.5, 3.0, and 4.5 mM, the cells underwent progressive growth suppression. With all four cell lines, growth suppression was increased by the combination of calcium and gadolinium over that seen in the presence of calcium alone. Gadolinium concentrations as low as 1–5 µM were effective. Increasing the calcium concentration from basal level to 1.5 mM also induced a strong differentiation response as indicated by a change in cell shape from round to flattened, by increased E-cadherin production, and by translocation of E-cadherin from the cytoplasm to the cell surface. Neither higher calcium concentrations (3.0 and 4.5 mM) nor combinations of calcium (3.0–4.5 mM) and gadolinium (50 µM) had additional effects on differentiation over that seen in the presence of 1.5 mM calcium alone. Taken together, these data indicate that the lanthanoid element can modulate growth without affecting differentiation.
Two questions are raised by this work. First is the potential to exploit these findings for improved growth control in the colon, and second is the mechanism(s) of enhanced growth inhibition in the presence of gadolinium. In regard to the first question, calcium (as noted above) has colon polyp chemopreventive activity, but alone, it is only modestly effective [18
]. Could a combination of calcium and gadolinium provide the basis for a more effective colon polyp chemoprevention strategy? This remains to be seen. A past study has shown that nifedipine, a calcium channel blocker used clinically in patients with cardiac disease, synergizes with calcium to induce apoptosis in the same colonic epithelial cells as used here [34
]. The clinically used calcium channel blockers have significant side effects in their own right and would be unlikely to be used systemically as part of a long-term cancer preventive strategy. The gastrointestinal tract, however, provides a unique opportunity for intervention without systemic exposure. This would be especially applicable for a cationic substance such as gadolinium that would be unlikely to cross the gastrointestinal barrier to become systemic. In support of such an approach, we have recently shown that a natural product containing calcium and a number of trace elements (including all of the naturally occurring lanthanoids) was more effective than calcium alone in suppressing outgrowth of adenomatous polyps in C57BL/6 mice on a high-fat diet over an 18-month period [19
]. We are currently assessing a combination of calcium and a mix of lanthanoids (formulated to include the same concentrations as found in the natural product) for colon polyp suppression in the same long-term animal model. Success in the animal model would support subsequent studies in human subjects at risk for polyp formation.
Gadolinium was examined here because this lanthanoid element has an orbital size and configuration similar to that of calcium but a higher overall charge density [20
]. While the current studies utilized gadolinium alone for “proof-of-concept,” other cationic metal ions, either alone or in combination, might prove to be more effective. This question would be difficult to address in an animal model, especially one that is inherently long term. However, the in vitro approach used in this manuscript should provide a way to assess multiple individual metals or combinations of metals for ability to synergize with calcium in suppression of colon epithelial cell growth. Currently, such studies are in process.
Regarding potential mechanisms of action, our studies showed that raising the calcium level to 1.5 mM induced differentiation (consistent with past reports [12
]). Raising the calcium further (with or without gadolinium) had no additional differentiation-inducing activity over that seen with calcium alone at 1.5 mM. In contrast, growth suppression increased with calcium alone up to 4.5 mM, and inclusion of gadolinium along with calcium enhanced growth inhibition at all three calcium concentrations (1.5, 3.0, and 4.5 mM). Gadolinium concentrations as low as 1–5 µM were effective. Growth-suppressing combinations of calcium and gadolinium induced cytotoxicity in a fraction of the cells. Calcium, by itself, participates in numerous signaling events that modulate cell proliferation and differentiation [12
] as well as cell death through both apoptotic and nonapoptotic mechanisms [33
for reviews]. Since in the absence of calcium, gadolinium alone had little effect on cell function, our hypothesis is that gadolinium acts in some way to sensitize the target cells to calcium. This is consistent with the known action of gadolinium on numerous calcium-regulatory molecules [22
CaSR is well established as a key calcium regulator in colonic epithelial cells. Our previous studies have demonstrated that calcium-induced growth suppression depends on the activation of CaSR [14
]. This is associated with differentiation and depends on a transient rise in the level of intracellular calcium [35
]. Based on these past data, we hypothesized that exposure of the colonic epithelial cells to gadolinium in conjunction with calcium would induce a rise in intracellular calcium over that seen with calcium alone. Our experiments failed to substantiate this, however. Quite the contrary, pretreatment with gadolinium reduced the subsequent influx of extracellular calcium that occurred in control cells exposed to 1.5 or 3.0 mM calcium. Ability of exogenous gadolinium to prevent extracellular calcium from entering the cell is consistent with calcium channel-blocking activity. Previous studies have shown that gadolinium interferes with receptor-gated calcium channels, as well as voltage-gated and mechanical stress-activated channels in a variety of cells [22
]. While some studies have suggested that limiting the rise in intracellular calcium might prevent apoptosis and promote cancer development, most studies have demonstrated that blocking calcium movement into the cell induces cell death [reviewed in 32
]. In the colon epithelial cells studied here, interference with calcium movement is clearly associated with reduced cell growth. That this relationship is cell specific is indicated by the almost contradictory findings in human dermal fibroblasts, where gadolinium is a potent growth inducer [29
In summary, a role for calcium in epithelial growth control is well established in the colon (as well as in other tissues). This is based on in vitro studies as well as on in vivo findings in both humans and in experimental animals. Calcium suppresses colonic epithelial cells, in part, by inducing a differentiation response. At higher calcium concentrations, processes that lead to cell death by apoptotic and nonapoptotic mechanisms are engaged. The present studies suggest that gadolinium enhances growth inhibitory/cytotoxic responses of colon epithelial cells to calcium but does not affect calcium-induced differentiation, per se. Whether the combination of calcium and gadolinium will, ultimately, prove to be useful as a colon cancer chemopreventive agent remains to be seen.